Solid forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid

ABSTRACT

The present invention relates to a substantially a solid form of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1, Solvate Form A and Compound 1, HCl Salt Form A), processes for making such forms, pharmaceutical compositions thereof, and methods of treatment therewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§119 to U.S. Provisional Application Ser. No. 61/321,729, filed Apr. 7,2010, and entitled “Solid Forms of3-(6-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid,” the entire contents of which is incorporatedherein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to solid state forms, for example,crystalline forms, of the compound3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid, pharmaceutical compositions thereof, and methods therewith.

BACKGROUND OF THE INVENTION

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a varietyof cell types, including absorptive and secretory epithelia cells, whereit regulates anion flux across the membrane, as well as the activity ofother ion channels and proteins. In epithelia cells, normal functioningof CFTR is critical for the maintenance of electrolyte transportthroughout the body, including respiratory and digestive tissue. CFTR iscomposed of approximately 1480 amino acids that encode a protein made upof a tandem repeat of transmembrane domains, each containing sixtransmembrane helices and a nucleotide binding domain. The twotransmembrane domains are linked by a large, polar, regulatory(R)-domain with multiple phosphorylation sites that regulate channelactivity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See, Gregory,R. J., et al. (1990) Nature 347:382-386; Rich, D. P., et al. (1990)Nature 347:358-362), (Riordan, J. R., et al. (1989) Science245:1066-1073). A defect in this gene causes mutations in CFTR resultingin cystic fibrosis (“CF”), the most common fatal genetic disease inhumans. Cystic fibrosis affects approximately one in every 2,500 infantsin the United States. Within the general United States population, up to10 million people carry a single copy of the defective gene withoutapparent ill effects. In contrast, individuals with two copies of the CFassociated gene suffer from the debilitating and fatal effects of CF,including chronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia lead to reduced apical anionsecretion, which causes an imbalance in ion and fluid transport. Theresulting decrease in anion transport contributes to enhanced mucusaccumulation in the lung and the accompanying microbial infections thatultimately cause death in CF patients. In addition to respiratorydisease, CF patients typically suffer from gastrointestinal problems andpancreatic insufficiency that, if left untreated, results in death. Inaddition, the majority of males with cystic fibrosis are infertile, andfertility is decreased among females with cystic fibrosis. In contrastto the severe effects of two copies of the CF associated gene,individuals with a single copy of the CF associated gene exhibitincreased resistance to cholera and to dehydration resulting fromdiarrhea—perhaps explaining the relatively high frequency of the CF genewithin the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease-causing mutations (Cutting, G. R., et al. (1990)Nature 346:366-369; Dean, M., et al. (1990) Cell 61:863:870; and Kerem,B-S., et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990)Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, more than 1000disease-causing mutations in the CF gene have been identified. The mostprevalent mutation is a deletion of phenylalanine at position 508 of theCFTR amino acid sequence, and is commonly referred to as ΔF508-CFTR.This mutation occurs in approximately 70 percent of the cases of cysticfibrosis and is associated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the endoplasmic reticulum (ER), and traffic to theplasma membrane. As a result, the number of channels present in themembrane is far less than observed in cells expressing wild-type CFTR.In addition to impaired trafficking, the mutation results in defectivechannel gating. Together, the reduced number of channels in the membraneand the defective gating lead to reduced anion transport acrossepithelia, leading to defective ion and fluid transport. (Quinton, P. M.(1990), FASEB J. 4: 2709-2727). Studies have shown, however, that thereduced numbers of ΔF508-CFTR in the membrane are functional, albeitless than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354:526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell.Biochem. 270: 12347-50). In addition to ΔF508-CFTR, other diseasecausing mutations in CFTR that result in defective trafficking,synthesis, and/or channel gating could be up- or down-regulated to alteranion secretion and modify disease progression and/or severity.

Although CFTR also transports a variety of molecules, it is clear thatthe transport of anions represents one element in an important mechanismof transporting ions and water across the epithelium. The other elementsinclude the epithelial Na⁺ channel, ENaC, Na⁺/2Cl⁻/K⁺ co-transporter,Na⁺—K⁺-ATPase pump, and the basolateral membrane K⁺ channels that areresponsible for the uptake of chloride into the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl-channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump, and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective endoplasmic reticulum (ER) processingof ABC transporters by the ER machinery has been shown to be theunderlying basis not only for CF disease, but for a wide range of otherisolated and inherited diseases. The two ways that the ER machinery canmalfunction are either by loss of coupling to ER export of the proteinsleading to degradation or by the ER accumulation of thesedefective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp745-751 (1999); Shastry, B. S., et al., Neurochem. International, 43, pp1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp 211-222(2002); Morello, J P et al., TIPS, 21, pp. 466-469 (2000); Bross P., etal., Human Mut., 14, pp. 186-198 (1999)].

The compound 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in salt form is disclosedin International PCT Publication WO 2007056341 (said publication beingincorporated herein by reference in its entirety) as a modulator of CFTRactivity and thus as a useful treatment for CFTR-mediated diseases suchas cystic fibrosis. Form I of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid (“Compound 1, Form I”), which is a substantially crystalline andsalt-free form, is disclosed in U.S. patent application Ser. No.12/327,902, filed Dec. 4, 2008, incorporated by reference herein in itsentirety. A need remains, however, for other stable solid forms of saidcompound that can be used readily in pharmaceutical compositionssuitable for use as therapeutics.

SUMMARY OF THE INVENTION

The present invention relates to solid polymorphic forms of the compound3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid (hereinafter “Compound 1”) which has the structure below:

Polymorphism is a known phenomena for solid forms of compounds. Thephysical characteristics of polymorphs are known to affect, for example,solubility, rate of dissolution, flow properties, rate of absorption,and stability. Hence, choice of a particular polymorph is important inthe development and preparation of compositions.

In one embodiment, Compound 1 is in solvated form, designated herein as“Compound 1, Solvate Form A.” In one embodiment, the solvate form isCompound 1, Methanol Solvate Form A. In another embodiment, the solvateis Compound 1, Ethanol Solvate Form A. Other embodiments include:

Compound 1, Acetone Solvate Form A;

Compound 1, 2-Propanol Solvate Form A;

Compound 1, Acetonitrile Solvate Form A;

Compound 1, Tetrahydrofuran Solvate Form A;

Compound 1, Methyl Acetate Solvate Form A;

Compound 1, 2-Butanone Solvate Form A;

Compound 1, Ethyl Formate Solvate Form A; and

Compound 1, 2-Methyltetrahydrofuran Solvate Form A.

In still another aspect, Compound 1 is in a salt form. The salt form isreferred to as “Compound 1, HCl Salt Form A”.

The Compound 1 solid forms disclosed herein and pharmaceuticalcompositions thereof are useful for lessening the severity ofCFTR-mediated diseases such as, for example, cystic fibrosis. TheCompound 1 solid forms can be used to prepare other Compound 1 solidforms, as well as pharmaceutical compositions comprising Compound 1solid forms, using the processes disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of Compound 1, SolvateForm A.

FIG. 2 provides X-ray diffraction patterns of Compound 1, Solvate Formsselected from:

1) Compound 1, Methanol Solvate Form A;

2) Compound 1, Ethanol Solvate Form A;

3) Compound 1, Acetone Solvate Form A;

4) Compound 1, 2-Propanol Solvate Form A;

5) Compound 1, Acetonitrile Solvate Form A;

6) Compound 1, Tetrahydrofuran Solvate Form A;

7) Compound 1, Methyl Acetate Solvate Form A;

8) Compound 1, 2-Butanone Solvate Form A;

9) Compound 1, Ethyl Formate Solvate Form A; and

10) Compound 1, 2-Methyltetrahydrofuran Solvate Form A.

FIG. 3 provides an X-ray diffraction pattern of Compound 1, MethanolSolvate Form A.

FIG. 4 provides an X-ray diffraction pattern of Compound 1, EthanolSolvate Form A.

FIG. 5 provides an X-ray diffraction pattern of Compound 1, AcetoneSolvate Form A.

FIG. 6 provides an X-ray diffraction pattern of Compound 1, 2-PropanolSolvate Form A.

FIG. 7 provides an X-ray diffraction pattern of Compound 1, AcetonitrileSolvate Form A.

FIG. 8 provides an X-ray diffraction pattern of Compound 1,Tetrahydrofuran Solvate Form A.

FIG. 9 provides an X-ray diffraction pattern of Compound 1, MethylAcetate Solvate Form A.

FIG. 10 provides an X-ray diffraction pattern of Compound 1, 2-ButanoneSolvate Form A.

FIG. 11 provides an X-ray diffraction pattern of Compound 1, EthylFormate Solvate Form A.

FIG. 12 provides an X-ray diffraction pattern of Compound 1,2-Methyltetrahydrofuran Solvate Form A.

FIG. 13 is a differential scanning calorimetry (DSC) trace of Compound1, Acetone Solvate Form A.

FIG. 14 is a Thermogravimetric analysis (TGA) plot of Compound 1,Acetone Solvate Form A.

FIG. 15 is a conformational image of Compound 1, Acetone Solvate Form Abased on single crystal X-ray analysis.

FIG. 16 is a conformational image of Compound 1, Solvate Form A based onsingle crystal X-ray analysis as a dimer.

FIG. 17 is a conformational image of Compound 1, Solvate Form A showinghydrogen bonding between carboxylic acid groups based on single crystalX-ray analysis.

FIG. 18 is a conformational image of Compound 1, Solvate Form A showingacetone as the solvate based on single crystal X-ray analysis.

FIG. 19 is a conformational image of the dimer of Compound 1, HCl SaltForm A.

FIG. 20 is a packing diagram of Compound 1, HCl Salt Form A.

FIG. 21 is an X-ray diffraction pattern of Compound 1, HCl Salt Form Acalculated from the crystal structure.

FIG. 22 is a solid state ¹³C NMR spectrum (15.0 kHz spinning) ofCompound 1, Acetone Solvate Form A.

FIG. 23 is a solid state ¹⁹F NMR spectrum (12.5 kHz spinning) ofCompound 1, Acetone Solvate Form A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/ last visited May 28, 2009,for CFTR mutations).

As used herein, “crystalline” refers to compounds or compositions wherethe structural units are arranged in fixed geometric patterns orlattices, so that crystalline solids have rigid long range order. Thestructural units that constitute the crystal structure can be atoms,molecules, or ions. Crystalline solids show definite melting points.

As used herein, the term “substantially crystalline” refers to a solidmaterial having predominantly long range order in the position of itsmolecules. For example, substantially crystalline materials have morethan about 85% crystallinity (e.g., more than about 90% crystallinity ormore than about 95% crystallinity). It is also noted that the term‘substantially crystalline’ includes the descriptor ‘crystalline,’ whichrefers to materials having 100% crystallinity.

As used herein, the term “modulating” means increasing or decreasing,e.g. activity, by a measurable amount.

As used herein, “isostructural solvate” refers to a compound crystallinelattice having a plurality of repeating cavities wherein some or all ofthe cavities may optionally be occupied by a solvent molecule which isthe same or different.

The term “DSC” means differential scanning calorimetry.

The term “TGA” means thermogravimetric analysis.

Compound 1, Solvate Form A

In one aspect, the invention features a Compound 1 solid form which isan isostructural solvate form, referred to as “Compound 1, Solvate FormA.”

Compound 1, Solvate Form A, as disclosed herein, comprises a crystallinelattice of Compound 1 in which voids in the crystalline lattice areempty, or occupied, or partially occupied by one or more molecules of asuitable solvent. Suitable solvents include, but are not limited to,methanol, ethanol, acetone, 2-propanol, acetonitrile, tetrahydrofuran,methyl acetate, 2-butanone, ethyl formate, and 2-methyl tetrahydrofuran.Certain physical characteristics of Compound 1 isostructural solvateforms, such as X-ray powder diffraction, melting point, and DSC, are notsubstantially affected by the particular solvent molecule in question.

In one embodiment, Compound 1, Solvate Form A is characterized by one ormore peaks at 21.50 to 21.90 degrees, 8.80 to 9.20 degrees, and 10.80 to11.20 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation.

In another embodiment, Compound 1, Solvate Form A is characterized byone or more peaks at 21.50 to 21.90 degrees, 8.80 to 9.20 degrees, 10.80to 11.20 degrees, 18.00 to 18.40 degrees, and 22.90 to 23.30 degrees inan X-ray powder diffraction obtained using Cu K alpha radiation.

In another embodiment, Compound 1, Solvate Form A is characterized byone or more peaks at 21.70, 8.98, and 11.04 degrees.

In another embodiment, Compound 1, Solvate Form A is characterized byone or more peaks at 21.70, 8.98, 11.04, 18.16, and 23.06 degrees.

In another embodiment, Compound 1, Solvate Form A is characterized by apeak at 21.50 to 21.90 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 21.70 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 8.80 to 9.20 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 8.98 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 10.80 to 11.20 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 11.04.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 18.00 to 18.40 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 18.16 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 22.90 to 23.30 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 23.06 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 20.40 to 20.80 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 20.63 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 22.00 to 22.40 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 22.22 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 18.40 to 18.80 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 18.57 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 16.50 to 16.90 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 16.66 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 19.70 to 20.10 degrees.

In another embodiment, Compound 1, Solvate Form A is furthercharacterized by a peak at 19.86 degrees.

In some embodiments, Compound 1, Solvate Form A is characterized by adiffraction pattern substantially similar to that of FIG. 1.

In some embodiments, Compound 1, Solvate Form A is characterized bydiffraction patterns substantially similar to those provided in FIG. 2.

In other embodiments, the solvate or solvate mixture that forms SolvateForm A with Compound 1 is selected from the group consisting of anorganic solvent of sufficient size to fit in the voids in thecrystalline lattice of Compound 1. In some embodiments, the solvate isof sufficient size to fit in voids measuring about 100 Å³.

In another embodiment, the solvate that forms Compound 1, Solvate Form Ais selected from the group consisting of methanol, ethanol, acetone,2-propanol, acetonitrile, tetrahydrofuran, methyl acetate, 2-butanone,ethyl formate, and 2-methyl tetrahydrofuran. Diffraction patterns areprovided for the following Compound 1, Solvate A forms: methanol (FIG.3), ethanol (FIG. 4), acetone (FIG. 5), 2-propanol (FIG. 6),acetonitrile (FIG. 7), tetrahydrofuran (FIG. 8), methyl acetate (FIG.9), 2-butanone (FIG. 10), ethyl formate (FIG. 11), and2-methyltetrahydrofuran (FIG. 12).

In another embodiment, the invention features crystalline Compound 1,Acetone Solvate Form A having a P2₁/n space group and the following unitcell dimensions: a=16.5235 (10) Å, b=12.7425 (8) Å, c=20.5512 (13) Å,α=90°, β=103.736 (4)°, and γ=90°.

In another embodiment, the invention provides Compound 1, Solvate Form Awhich exhibits two or more phase transitions as determined by DSC or asimilar analytic method known to the skilled artisan. In someembodiments, the DSC of Compound 1, Solvate Form A is substantiallysimilar to the DSC trace depicted in FIG. 13.

In another embodiment of this aspect, the DSC gives two phasetransitions.

In another embodiment, the DSC gives three phase transitions.

In another embodiment, one of the phase transitions occurs between 200and 207° C.

In another embodiment, one of the phase transitions occurs between 204and 206° C.

In another embodiment, one of the phase transitions occurs between 183and 190° C.

In another embodiment, one of the phase transitions occurs between 185and 187° C.

In another embodiment, the melting point of Compound 1, Solvate Form Ais between 183° C. to 190° C.

In another embodiment, the melting point of Compound 1, Solvate Form Ais between 185° C. to 187° C.

In another embodiment, Compound 1, Solvate Form A comprises 1 to 10weight percent (wt. %) solvate as determined by TGA. In someembodiments, the TGA of Compound 1, Solvate Form A is substantiallysimilar to the TGA trace depicted in FIG. 14.

In another embodiment, Compound 1, Solvate Form A comprises 2 to 5 wt. %solvate as determined by TGA or a similar analytic method known to theskilled artisan.

In another embodiment, the conformation of Compound 1, Acetone SolvateForm A is substantially similar to that depicted in FIG. 15, which isbased on single X-ray analysis.

In another embodiment, Compound 1, Acetone Solvate Form A has a P2₁/nspace group, and the following unit cell dimensions:

a=16.5235 (10) Å α=90°

b=12.7425 (8) Å β=103.736 (4)°

c=20.5512 (13) Å γ=90°.

Compound 1, HCl Salt Form A

In another aspect, the invention provides another Compound 1 solid formwhich is a crystalline HCl salt. This solid form is designated asCompound 1, HCl Salt Form A.

In one embodiment, Compound 1, HCl Salt Form A is characterized by oneor more peaks at 8.80 to 9.20 degrees, 17.30 to 17.70 degrees, and 18.20to 18.60 degrees in an X-ray powder diffraction obtained using Cu Kalpha radiation.

In another embodiment, Compound 1, HCl Salt Form A is characterized byone or more peaks at 8.80 to 9.20 degrees, 17.30 to 17.70 degrees, 18.20to 18.60 degrees, 10.10 to 10.50, and 15.80 to 16.20 degrees in an X-raypowder diffraction obtained using Cu K alpha radiation.

In another embodiment, Compound 1, HCl Salt Form A is characterized byone or more peaks at 8.96, 17.51, and 18.45 degrees.

In another embodiment, Compound 1, HCl Salt Form A is characterized byone or more peaks at 8.96, 17.51, 18.45. 10.33, and 16.01 degrees.

In another embodiment, Compound 1, HCl Salt Form A is characterized by apeak at 8.80 to 9.20 degrees.

In another embodiment, Compound 1, HCl Salt Form A is characterized by apeak at 8.96 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 17.30 to 17.70 degrees.

In another embodiment, Compound 1, HCl Salt Form A is characterized by apeak at 17.51 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 18.20 to 18.60 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 18.45 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 10.10 to 10.50 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 10.33 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 15.80 to 16.20 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 16.01 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 11.70 to 12.10 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 11.94 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 7.90 to 8.30 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 8.14 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 9.90 to 10.30 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 10.10 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 16.40 to 16.80 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 16.55 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 9.30 to 9.70 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 9.54 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 16.40 to 16.80 degrees.

In another embodiment, Compound 1, HCl Salt Form A is furthercharacterized by a peak at 16.55 degrees.

In some embodiments, Compound 1, HCl Salt Form A is characterized as adimer, as depicted in FIG. 19.

In some embodiments, Compound 1, HCl Salt Form A is characterized by thepacking diagram depicted in FIG. 20.

In some embodiments, Compound 1, HCl Salt Form A is characterized by adiffraction pattern substantially similar to that of FIG. 21.

In another embodiment, the invention features crystalline Compound 1,HCl Salt Form A having a P⁻1 space group and the following unit celldimensions: a=10.2702 (2) Å, b=10.8782 (2) Å, c=12.4821 (3) Å, α=67.0270(10)°, β=66.1810 (10)°, and γ=72.4760 (10)°.

In another embodiment, the invention features a kit comprising Compound1, Solvate Form A or Compound 1, HCl Salt Form A and instructions foruse thereof.

Synthesis of Compound 1, Solvate Form A and Compound 1, HCl Salt Form A

The solid forms of the invention designated and described above asCompound 1, Solvate Form A and Compound 1, HCl Salt Form A, can beprepared from precursors including Compound 1 and from other solid formsof Compound 1. This section describes the synthesis of Compound 1 andother solid forms of Compound 1, as well as the conversion of theseother solid forms to the solid forms of the invention designated asCompound 1, Solvate Form A and Compound 1, HCl Salt Form A.

Scheme 1 depicts the preparation of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride,which is used to make Compound 1 (See Scheme 4). The starting material,2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid, is commerciallyavailable from Saltigo (an affiliate of the Lanxess Corporation).Reduction of the carboxylic acid moiety in2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid provides the primaryalcohol, which is converted to the corresponding primary alkyl chloride5-(chloromethyl)-2,2-difluorobenzo[d][1,3]dioxole using thionylchloride. The alkyl chloride is subsequently converted to the nitrile2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile by displacementusing sodium cyanide. Treatment of the nitrile with1-bromo-2-chloroethane in the presence of base provides1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile.Conversion of the nitrile to1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acidunder basic conditions, followed by treatment with thionyl chloride,provides the requisite acid chloride.

Scheme 2 provides an alternative synthesis of the requisite acidchloride. The compound 5-bromomethyl-2,2-difluoro-1,3-benzodioxole iscoupled with ethyl cyanoacetate in the presence of a palladium catalystto form the corresponding alpha cyano ethyl ester. Saponification of theester moiety to the carboxylic acid gives the cyanoethyl compound.Alkylation of the cyanoethyl compound with 1-bromo-2-chloro ethane inthe presence of base gives the cyanocyclopropyl compound. Treatment ofthe cyanocyclopropyl compound with base gives the carboxylate salt,which is converted to the carboxylic acid by treatment with acid.Conversion of the carboxylic acid to the acid chloride is thenaccomplished using a chlorinating agent such as thionyl chloride or thelike.

Scheme 3 depicts the preparation of tert-butyl3-(6-amino-3-methylpyridin-2-yl)benzoate, which is coupled with1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride(See Schemes 1 and 2) in Scheme 4 to give Compound 1 as an acid salt.Palladium-catalyzed coupling of 2-bromo-3-methylpyridine with3-(tert-butoxycarbonyl)phenylboronic acid gives tert-butyl3-(3-methylpyridin-2-yl)benzoate, which is subsequently converted via aseries of steps to the desired compound.

Compound 1 is prepared as an acid salt as provided in Scheme 4. Couplingthe acid chloride of Schemes 1 or 2 with the amine of Scheme 3 usingtriethyl amine and catalytic 4-dimethylaminopyridine or the likeinitially provides the tert-butyl ester3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate,which is the tert-butyl ester precursor of Compound 1. Treatment of thetert-butyl ester with an acid, such as HCl, gives the HCl salt ofCompound 1. The HCl salt of Compound 1, which is typically a crystallinesolid, can be used to prepare other solid forms of Compound 1, includingthe solid forms of the invention, Compound 1, Solvate From A andCompound 1, HCl Salt Form A.

Preparation of Compound 1, Solvate Form A from the HCl Salt of Compound1

Compound 1, Solvate Form A can be prepared by dispersing or dissolving asalt form, such as the HCl salt of Compound 1, in an appropriate solventfor an effective amount of time, followed by isolation of crystallineCompound 1, Solvate Form A by filtration.

Preparation of Compound 1, HCl Salt Form A from the HCl Salt of Compound1

Compound 1, HCl Salt Form A can be prepared from the HCl salt ofCompound 1 by dissolving the HCl salt of Compound 1 in a minimum ofsolvent and removing the solvent by slow evaporation. In an embodiment,the solvent is an alcohol. In another embodiment, the solvent isethanol. Slow evaporation is generally carried out by impeding theevaporation of the solvent. For example, in one embodiment, slowevaporation involves dissolving the HCl salt of Compound 1 in a vial andcovering the vial with parafilm that contains a hole poked in it.

Preparation of Compound 1, Solvate Form A from Compound 1, Form I

Compound 1, Form 1 (described above and as disclosed in U.S. patentapplication Ser. No. 12/327,902, filed Dec. 4, 2008) is another solidform of Compound 1 that can be used to prepare the solid forms of theinvention, particularly Compound 1, Solvate Form A.

Compound 1, Form 1 can be prepared from the HCl salt of Compound 1 bydispersing or dissolving the HCl salt in an appropriate solvent for aneffective amount of time. The appropriate solvent may be water or analcohol/water mixture, such as a 50% methanol/water mixture, even thoughthe HCl salt form of Compound 1 is only sparingly soluble in water.Alternatively, the appropriate solvent is water.

Other acid salts of Compound 1 may also be used to prepare Compound 1,Form 1, such as, for example, salts derived from other mineral ororganic acids. The other salts also result from acid-mediated hydrolysisof the tert-butyl ester precursor of Compound 1 (See Scheme 4). Saltsderived from other acids may include, for example, nitric, sulfuric,phosphoric, boric, acetic, benzoic, and malonic. These salt forms ofCompound 1 may or may not be soluble, depending upon the solvent used,but lack of solubility does not hinder formation of Compound 1, Form I.The effective amount of time for formation of Compound 1, Form I from asalt of Compound 1 can be any time between 2 and 24 hours or greater. Itis recognized that the amount of time needed is inversely proportionalto the temperature. That is, the higher the temperature, the less timeneeded to affect dissociation of acid to form Compound 1, Form I. Whenthe solvent is water, stifling the dispersion for approximately 24 hoursat room temperature provides Form I in an approximately 98% yield. If asolution of the salt of Compound 1 is desired for process purposes, anelevated temperature may be used. After stirring the solution for aneffective amount of time at the elevated temperature, recrystallizationupon cooling provides substantially pure Compound 1, Form I.“Substantially pure” refers to greater than about 90% purity, or greaterthan about 95% purity, or greater than about 98% purity, or greater thanabout 99% purity. The temperature selected depends in part on thesolvent used and is well within the determination capabilities of one ofordinary skill in the art and is typically between room temperature andabout 80° C.

Alternatively, Compound 1, Form I can be formed directly from thetert-butyl ester precursor of Compound 1 (See Scheme 4) by treatmentwith an appropriate acid, such as, for example, formic acid, underappropriate reaction conditions to give Compound 1, Form I. For example,the tert-butyl ester is reacted with an appropriate acid, such as formicacid, at 60 to 80° C. for 7 to 9 hours before cooling to ambienttemperatures, adding the reaction mixture to water, and reheating to 60to 80° C. for an effective amount of time. Compound 1, Form 1 is thenisolated by filtration.

Compound 1, Form I may be further purified by recrystallization from anorganic solvent. Examples of organic solvents include, but are notlimited to, toluene, cumene, anisol, 1-butanol, isopropyl acetate, butylacetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketone,and 1-propanol-water mixtures. The temperature may be as describedabove. For example, Compound 1, Form I is dissolved in 1-butanol at 75°C. until it is completely dissolved. Cooling down the solution to 10° C.at a rate of 0.2° C./min yields crystals of Compound 1, Form I which maybe isolated by filtration.

As indicated, Compound 1, Form 1 can be used to prepare Compound 1,Solvate Form A. Accordingly, an amount of Compound 1, Form I is slurriedin an appropriate solvent at a sufficient concentration for a sufficienttime. The slurry is then filtered centrifugally or under vacuum anddried at ambient conditions for sufficient time to yield Compound 1,Solvate Form A.

In some embodiments, about 20 to 40 mg of Compound 1, Form I is slurriedin about 400 to 600 μL of an appropriate solvent. In another embodiment,about 25 to 35 mg of Compound 1, Form I is slurried in about 450 to 550μL of an appropriate solvent. In another embodiment, about 30 mg ofCompound 1, Form I is slurried in about 500 μL of an appropriatesolvent.

In some embodiments, the time that Compound 1, Form I is allowed toslurry with the solvent is between 1 hour and four days. Moreparticularly, the time that Compound 1, Form I is allowed to slurry withthe solvent is between 1 and 3 days. More particularly, the time is 2days.

In some embodiments, the appropriate solvent is selected from an organicsolvent of sufficient size to fit the voids in the crystalline latticeof Compound 1. In other embodiments, the solvate is of sufficient sizeto fit in voids measuring about 100 Å³.

In other embodiments, the solvent is selected from the group consistingof methanol, ethanol, acetone, 2-propanol, acetonitrile,tetrahydrofuran, methyl acetate, 2-butanone, ethyl formate, and 2-methyltetrahydrofuran.

In other embodiments, a mixture of two or more of these solvents may beused to obtain Compound 1, Solvate Form A. Alternatively, Compound 1,Solvate Form A may be obtained from a mixture comprising one or more ofthese solvents and water.

In some embodiments, the effective amount of time for drying Compound 1,Solvate Form A is 1 to 24 hours. More particularly, the time is 6 to 18hours. More particularly, the time is about 12 hours.

Uses, Compositions and Administration

Pharmaceutically Acceptable Compositions

In another aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise Compound1, Solvate Form A or Compound 1, HCl Salt Form A, as described herein,and optionally comprise a pharmaceutically acceptable carrier, adjuvant,or vehicle. In certain embodiments, these compositions optionallyfurther comprise one or more additional therapeutic agents.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers; alumina; aluminumstearate; lecithin; serum proteins, such as human serum albumin; buffersubstances, such as phosphates, glycine, sorbic acid, or potassiumsorbate; partial glyceride mixtures of saturated vegetable fatty acids;water; salts, or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, or magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blockpolymers; wool fat; sugars, such as lactose, glucose, or sucrose;starches, such as corn starch and potato starch; cellulose and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose,and cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients, such as cocoa butter and suppository waxes; oils, such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil, or soybean oil; glycols, such as propylene glycol or polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; and phosphate buffer solutions, as well as other non-toxiccompatible lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, releasing agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the composition, according to thejudgment of the formulator.

Methods of Treatment

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by CFTR. Incertain embodiments, the present invention provides a method of treatinga condition, disease, or disorder implicated by a deficiency of CFTRactivity, the method comprising administering a composition comprising asolid state form of Compound 1 selected from Compound 1, Solvate Form Aand Compound 1, HCl Salt Form A, described herein, to a subject,preferably a mammal, in need thereof.

As used herein, a “CFTR-mediated disease” is a disease selected fromcystic fibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, I-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders, Huntington's, spinocerebullar ataxia type I,spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,myotonic dystrophy, spongiform encephalopathies, hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eye disease,Sjogren's disease, Osteoporosis, Osteopenia, Gorham's Syndrome, chloridechannelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), inherited disorders of the structure and/orfunction of cilia, PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, or ciliary aplasia.

In certain embodiments, the present invention provides a method oftreating a CFTR-mediated disease in a human comprising the step ofadministering to said human an effective amount of a compositioncomprising Compound 1, Solvate Form A or Compound 1, HCl Salt Form A,described herein.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis in a human comprising thestep of administering to said human a composition comprising Compound 1,Solvate Form A or Compound 1, HCl Salt Form A described herein.

In another embodiment, said human has cystic fibrosis transmembranereceptor (CFTR) with a ΔF508 mutation. In another embodiment, said humanhas cystic fibrosis transmembrane receptor (CFTR) with a R117H mutation.In another embodiment, said human has cystic fibrosis transmembranereceptor (CFTR) with a G551D mutation.

According to the invention, an “effective amount” of Compound 1, SolvateForm A or a pharmaceutically acceptable composition thereof is thatamount effective for treating or lessening the severity of any of thediseases recited above.

Compound 1, Solvate Form A or Compound 1, HCl Salt Form A, orpharmaceutically acceptable compositions thereof, may be administeredusing any amount and any route of administration effective for treatingor lessening the severity of one or more of the diseases recited above.

In certain embodiments, Compound 1, Solvate Form A or Compound 1, HClSalt Form A, as described herein, or pharmaceutically acceptablecompositions thereof, are useful for treating or lessening the severityof cystic fibrosis in patients who exhibit residual CFTR activity in theapical membrane of respiratory and non-respiratory epithelia. Thepresence of residual CFTR activity at the epithelial surface can bereadily detected using methods known in the art, e.g., standardelectrophysiological, biochemical, or histochemical techniques. Suchmethods identify CFTR activity using in vivo or ex vivoelectrophysiological techniques, measurement of sweat or salivary Cl⁻concentrations, or ex vivo biochemical or histochemical techniques tomonitor cell surface density. Using such methods, residual CFTR activitycan be readily detected in patients heterozygous or homozygous for avariety of different mutations, including patients homozygous orheterozygous for the most common mutation, ΔF508.

In one embodiment, Compound 1, Solvate Form A or Compound 1, HCl SaltForm A, as described herein, or pharmaceutically acceptable compositionsthereof, are useful for treating or lessening the severity of cysticfibrosis in patients within certain genotypes exhibiting residual CFTRactivity, e.g., class III mutations (impaired regulation or gating),class IV mutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Transmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, Compound 1, Solvate Form A or Compound 1, HCl SaltForm A, as described herein, or pharmaceutically acceptable compositionsthereof, are useful for treating or lessening the severity of cysticfibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic insufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form,” as used herein, refers to aphysically discrete unit of agent appropriate for the patient to betreated. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific effective dose level for any particular patient or organismwill depend upon a variety of factors, including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient,” or“subject,” as used herein, means an animal, preferably a mammal, andmost preferably a human.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg, and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

In certain embodiments, the dosage amount of Compound 1, Solvate Form Aor Compound 1, HCl Salt Form A are in the dosage unit is from 100 mg to1,000 mg. In another embodiment, the dosage amount of Compound 1,Solvate Form A is from 200 mg to 900 mg. In another embodiment, thedosage amount of Compound 1, Solvate Form A or Compound 1, HCl Salt FormA is from 300 mg to 800 mg. In another embodiment, the dosage amount ofCompound 1, Solvate Form A or Compound 1, HCl Salt Form A is from 400 mgto 700 mg. In another embodiment, the dosage amount of Compound 1,Solvate Form A or Compound 1, HCl Salt Form A is from 500 mg to 600 mg.

In another embodiment, the present invention comprises jet millingCompound 1, Solvate Form A or Compound 1, HCl Salt Form A in a suitable,conventional milling apparatus using air pressure suitable to produceparticles having a significant particle size fraction between 0.1microns and 50 microns. In another embodiment, the particle size isbetween 0.1 microns and 20 microns. In another embodiment, the particlessize is between 0.1 microns and 10 microns. In another embodiment, theparticle size is between 1.0 microns and 5 microns. In still anotherembodiment, Compound 1, Solvate Form A or Compound 1, HCl Salt Form Ahas a particle size D50 of 2.0 microns.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed, including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions, which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing Compound 1, Solvate Form Aor Compound 1, HCL Salt Form A with suitable non-irritating excipientsor carriers such as cocoa butter, polyethylene glycol or a suppositorywax which are solid at ambient temperature but liquid at bodytemperature and therefore melt in the rectum or vaginal cavity andrelease the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the Compound1, Solvate Form A or Compound 1, HCL Salt Form A is mixed with at leastone inert, pharmaceutically acceptable excipient or carrier such assodium citrate or dicalcium phosphate and/or a) fillers or extenderssuch as starches, lactose, sucrose, glucose, mannitol, and silicic acid,b) binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants suchas glycerol, d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, e) solution retarding agents such as paraffin, f)absorption accelerators such as quaternary ammonium compounds, g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, h) absorbents such as kaolin and bentonite clay, and i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets and pills, the dosage form may alsocomprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets, and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

It will also be appreciated that Compound 1, Solvate Form A or Compound1, HCl Salt Form A as described herein or pharmaceutically acceptablecompositions thereof can be employed in combination therapies, that is,Compound 1, Solvate Form A or Compound 1, HCl Salt Form A can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with another agentused to treat the same disorder), or they may achieve different effects(e.g., control of any adverse effects). As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In one embodiment, the additional agent is(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.In another embodiment, the additional agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another embodiment, the additional agent is selected from Table 1:

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

In another embodiment, the additional agent is any combination of theabove agents. For example, the composition may comprise Compound 1,(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide,andN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another example, the composition may comprise Compound 1,N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide,and any one of the compounds from Table 1, i.e. compounds 1 through 14of Table 1, or any combination thereof.

In one embodiment, the additional therapeutic agent is an antibiotic.Exemplary antibiotics useful herein include tobramycin, includingtobramycin inhaled powder (TIP), azithromycin, aztreonam, including theaerosolized form of aztreonam, amikacin, including liposomalformulations thereof, ciprofloxacin, including formulations thereofsuitable for administration by inhalation, levoflaxacin, includingaerosolized formulations thereof, and combinations of two antibiotics,e.g., fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodilators include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan Compound 1, Solvate Form A or Compound 1, HCl Salt Form A, i.e., anagent that has the effect of modulating CFTR activity. Exemplary suchagents include ataluren (“PTC124®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), and cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid).

In another embodiment, the additional agent is a nutritional agent.Exemplary nutritional agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, amrinone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound disclosed inWO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740.

In another embodiment, the additional agent is a benzo(c)quinoliziniumderivative that exhibits CFTR modulation activity or a benzopyranderivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed inU.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.

In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256,WO2006127588, or WO2007044560.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Compound 1, Solvate Form A or Compound 1, HCl Salt Form A, as describedherein, or pharmaceutically acceptable compositions thereof, may also beincorporated into compositions for coating an implantable medicaldevice, such as prostheses, artificial valves, vascular grafts, stents,and catheters. Accordingly, the present invention, in another aspect,includes a composition for coating an implantable device comprisingCompound 1, Solvate Form A or Compound 1, HCl Salt Form A, as describedherein, or pharmaceutically acceptable compositions thereof, and inclasses and subclasses herein, and a carrier suitable for coating saidimplantable device. In still another aspect, the present inventionincludes an implantable device coated with a composition comprisingCompound 1, Solvate Form A or Compound 1, HCl Salt Form A as describedherein, or pharmaceutically acceptable compositions thereof, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. Optionally, thecoatings may be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids, or combinationsthereof to impart controlled release characteristics in the composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Methods & Materials

Differential Scanning Calorimetry (DSC)

The Differential scanning calorimetry (DSC) data for Compound 1, SolvateForm A were collected using a DSC Q100 V9.6 Build 290 (TA Instruments,New Castle, Del.). Temperature was calibrated with indium, and heatcapacity was calibrated with sapphire. Samples of 3-6 mg were weighedinto aluminum pans that were crimped using lids with 1 pin hole. Thesamples were scanned from 25° C. to 350° C. at a heating rate of 1.0°C./min and with a nitrogen gas purge of 50 ml/min. Data were collectedby Thermal Advantage Q Series™ version 2.2.0.248 software and analyzedby Universal Analysis software version 4.1D (TA Instruments, New Castle,Del.). The reported numbers represent single analyses.

Jet Milling Description

Unmicronized Compound 1, Solvate Form A or Compound 1, HCl Salt Form Ais sieved to de-lump it prior to placing it into the jet mill hopper.All sieves are disposable and received a wipe prior to use. UnmicronizedCompound 1, Solvate Form A or Compound 1, HCl Salt Form A is added tothe jet mill hopper at a controlled feeding rate using compressednitrogen gas. The gas pressure range is 40-45/45-70 (Venturi/Mill) PSIand the feeding rate range is 0.5-1.6 Kg/Hour. The Compound 1, SolvateForm A or Compound 1, HCl Salt Form A is micronized in the mill throughparticle-particle and particle-wall collisions and the processedCompound 1, Solvate Form A or Compound 1, HCl Salt Form A is emptiedinto the micronized product containers. It is believed that one ofordinary skill in the art may also achieve Compound 1, Solvate Form A orCompound 1, HCl Salt Form A with a favorable particle size through pinmilling based in part on the conditions described above.

XRPD (X-Ray Powder Diffraction)

X-Ray diffraction (XRD) data were collected on either a Bruker D8DISCOVER or Bruker APEX II powder diffractometer. The Bruker D8 DISCOVERDiffractomer with HI-STAR 2-dimensional detector and a flat graphitemonochromator. Cu sealed tube with K-alpha radiation was used at 40 kV,35 mA. The samples were placed on zero-background silicon wafers at 25°C. For each sample, two data frames were collected at 120 seconds eachat 2 different θ₂ angles: 8° and 26°. The data were integrated withGADDS software and merged with DIFFRACT^(plus)EVA software.Uncertainties for the reported peak positions are ±0.2 degrees. equippedwith sealed tube Cu Kα source and an Apex II CCD detector.

The Bruker II powder diffractomer was equipped with a sealed tube CuKsource and an APEX II CCD detector. Structures were solved and refinedusing the SHELX program. (Sheldrick, G. M., Acta Cryst. (2008) A64,112-122).

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals.

2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchased fromSaltigo (an affiliate of the Lanxess Corporation).

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) was slurried in toluene (10 vol). Vitride® (2 eq) was added viaaddition funnel at a rate to maintain the temperature at 15 to 25° C. Atthe end of the addition, the temperature was increased to 40° C. for 2hours (h), then 10% (w/w) aqueous (aq) NaOH (4.0 eq) was carefully addedvia addition funnel, maintaining the temperature at 40 to 50° C. Afterstirring for an additional 30 minutes (min), the layers were allowed toseparate at 40° C. The organic phase was cooled to 20° C., then washedwith water (2×1.5 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that was useddirectly in the next step.

Preparation of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) was dissolved inMTBE (5 vol). A catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP)(1 mol %) was added and SOCl₂ (1.2 eq) was added via addition funnel.The SOCl₂ was added at a rate to maintain the temperature in the reactorat 15 to 25° C. The temperature was increased to 30° C. for 1 hour, andthen was cooled to 20° C. Water (4 vol) was added via addition funnelwhile maintaining the temperature at less than 30° C. After stirring foran additional 30 minutes, the layers were allowed to separate. Theorganic layer was stirred, and 10% (w/v) aq NaOH (4.4 vol) was added.After stirring for 15 to 20 minutes, the layers were allowed toseparate. The organic phase was then dried (Na₂SO₄), filtered, andconcentrated to afford crude5-chloromethyl-2,2-difluoro-1,3-benzodioxole that was used directly inthe next step.

Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) was added to a slurry of NaCN (1.4 eq) in DMSO (3 vol),while maintaining the temperature between 30 to 40° C. The mixture wasstirred for 1 hour, and then water (6 vol) was added, followed by methyltert-butyl ether (MTBE) (4 vol). After stirring for 30 minutes, thelayers were separated. The aqueous layer was extracted with MTBE (1.8vol). The combined organic layers were washed with water (1.8 vol),dried (Na₂SO₄), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) that was useddirectly in the next step.

Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile

A reactor was purged with nitrogen and charged with toluene (900 mL).The solvent was degassed via nitrogen sparge for no less than 16 hours.To the reactor was then charged Na₃PO₄ (155.7 g, 949.5 mmol), followedby bis(dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A 10%w/w solution of tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) wascharged over 10 minutes at 23° C. from a nitrogen purged additionfunnel. The mixture was allowed to stir for 50 minutes, at which time5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added over1 minute. After stirring for an additional 50 minutes, the mixture wascharged with ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 minutes,followed by water (4.5 mL) in one portion. The mixture was heated to 70°C. over 40 minutes and analyzed by HPLC every 1 to 2 hours for thepercent conversion of the reactant to the product. After completeconversion was observed (typically 100% conversion after 5 to 8 hours),the mixture was cooled to 20 to 25° C. and filtered through a celitepad. The celite pad was rinsed with toluene (2×450 mL), and the combinedorganics were concentrated to 300 mL under vacuum at 60 to 65° C. Theconcentrate was charged with DMSO (225 mL) and concentrated under vacuumat 70 to 80° C. until active distillation of the solvent ceased. Thesolution was cooled to 20 to 25° C. and diluted to 900 mL with DMSO inpreparation for Step 2. ¹H NMR (500 MHz, CDCl₃) δ 7.16-7.10 (m, 2H),7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H), 4.19 (m, 2H), 1.23 (t, J=7.1 Hz,3H).

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

The DMSO solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile fromabove was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20 minuteswhile maintaining an internal temperature less than 40° C. The mixturewas then heated to 75° C. over 1 hour and analyzed by HPLC every 1 to 2hour for percent conversion. When a conversion of greater than 99% wasobserved (typically after 5 to 6 hours), the reaction was cooled to 20to 25° C. and extracted with MTBE (2×525 mL), with sufficient time toallow for complete phase separation during the extractions. The combinedorganic extracts were washed with 5% NaCl (2×375 mL). The solution wasthen transferred to equipment appropriate for a 1.5 to 2.5 Torr vacuumdistillation that was equipped with a cooled receiver flask. Thesolution was concentrated under vacuum at less than 60° C. to remove thesolvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was thendistilled from the resulting oil at 125 to 130° C. (oven temperature)and 1.5 to 2.0 Torr. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrilewas isolated as a clear oil in 66% yield from5-bromo-2,2-difluoro-1,3-benzodioxole (2 steps) and with an HPLC purityof 91.5% AUC (corresponds to a w/w assay of 95%). ¹H NMR (500 MHz, DMSO)δ 7.44 (br s, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.22 (dd, J=8.2, 1.8 Hz, 1H),4.07 (s, 2H).

Preparation of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile

A mixture of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (1.0 eq),50 wt % aqueous KOH (5.0 eq) 1-bromo-2-chloroethane (1.5 eq), andOct₄NBr (0.02 eq) was heated at 70° C. for 1 hour. The reaction mixturewas cooled, then worked up with MTBE and water. The organic phase waswashed with water and brine. The solvent was removed to afford(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid

(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile washydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80° C.overnight. The mixture was cooled to room temperature, and the ethanolwas evaporated under vacuum. The residue was taken up in water and MTBE,1 M HCl was added, and the layers were separated. The MTBE layer wasthen treated with dicyclohexylamine (DCHA) (0.97 equiv). The slurry wascooled to 0° C., filtered, and washed with heptane to give thecorresponding DCHA salt. The salt was taken into MTBE and 10% citricacid and stirred until all the solids had dissolved. The layers wereseparated, and the MTBE layer was washed with water and brine. A solventswap to heptane followed by filtration gave1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid afterdrying in a vacuum oven at 50° C. overnight.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonyl chloride

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.2eq) was slurried in toluene (2.5 vol), and the mixture was heated to 60°C. SOCl₂ (1.4 eq) was added via addition funnel. The toluene and SOCl₂were distilled from the reaction mixture after 30 minutes. Additionaltoluene (2.5 vol) was added, and the resulting mixture was distilledagain, leaving the product acid chloride as an oil, which was usedwithout further purification.

Preparation of tert-butyl-3-(3-methylpyridin-2-yl)benzoate

2-Bromo-3-methylpyridine (1.0 eq) was dissolved in toluene (12 vol).K₂CO₃ (4.8 eq) was added, followed by water (3.5 vol). The resultingmixture was heated to 65° C. under a stream of N₂ for 1 hour.3-(t-Butoxycarbonyl)phenylboronic acid (1.05 eq) and Pd(dppf)Cl₂.CH₂Cl₂(0.015 eq) were then added, and the mixture was heated to 80° C. After 2hours, the heat was turned off, water was added (3.5 vol), and thelayers were allowed to separate. The organic phase was then washed withwater (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2eq MsOH, 7.7 vol). The aqueous phase was made basic with 50% aqueousNaOH (2 eq) and extracted with EtOAc (8 vol). The organic layer wasconcentrated to afford crude tert-butyl-3-(3-methylpyridin-2-yl)benzoate(82%) that was used directly in the next step.

Preparation of2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide

tert-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) was dissolved inEtOAc (6 vol). Water (0.3 vol) was added, followed by urea-hydrogenperoxide (3 eq). Phthalic anhydride (3 eq) was then added portionwise tothe mixture as a solid at a rate to maintain the temperature in thereactor below 45° C. After completion of the phthalic anhydrideaddition, the mixture was heated to 45° C. After stirring for anadditional 4 hours, the heat was turned off. 10% w/w aqueous Na₂SO₃ (1.5eq) was added via addition funnel. After completion of Na₂SO₃ addition,the mixture was stirred for an additional 30 minutes, and the layersseparated. The organic layer was stirred, and 10% wt/wt aqueous. Na₂CO₃(2 eq) was added. After stirring for 30 minutes, the layers were allowedto separate. The organic phase was washed 13% w/v aq NaCl. The organicphase was then filtered and concentrated to afford crude2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (95%) thatwas used directly in the next step.

Preparation of tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate

A solution of 2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide(1 eq) and pyridine (4 eq) in acetonitrile (8 vol) was heated to 70° C.A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) wasadded over 50 minutes via addition funnel while maintaining thetemperature at less than 75° C. The mixture was stirred for anadditional 0.5 hours after complete addition. The mixture was thenallowed to cool to ambient temperature. Ethanolamine (10 eq) was addedvia addition funnel. After stirring for 2 hours, water (6 vol) wasadded, and the mixture was cooled to 10° C. After stirring for 3 hours,the solid was collected by filtration and washed with water (3 vol), 2:1acetonitrile/water (3 vol), and acetonitrile (2×1.5 vol). The solid wasdried to constant weight (less than 1% difference) in a vacuum oven at50° C. with a slight N₂ bleed to affordtert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate as a red-yellowsolid (53% yield).

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate

The crude acid chloride described above was dissolved in toluene (2.5vol based on acid chloride) and added via addition funnel to a mixtureof tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate (1 eq), DMAP,(0.02 eq), and triethylamine (3.0 eq) in toluene (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2 hours,water (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) was added to thereaction mixture. After stirring for 30 minutes, the layers wereseparated. The organic phase was then filtered and concentrated toafford a thick oil of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(quantitative crude yield). Acetonitrile (3 vol based on crude product)was added and distilled until crystallization occurred. Water (2 volbased on crude product) was added, and the mixture stirred for 2 hours.The solid was collected by filtration, washed with 1:1 (by volume)acetonitrile/water (2×1 volumes based on crude product), and partiallydried on the filter under vacuum. The solid was dried to a constantweight (less than 1% difference) in a vacuum oven at 60° C. with aslight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoateas a brown solid.

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCL salt

To a slurry of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(1.0 eq) in MeCN (3.0 vol) was added water (0.83 vol) followed byconcentrated aqueous HCl (0.83 vol). The mixture was heated to 45±5° C.After stirring for 24 to 48 hours, the reaction was complete, and themixture was allowed to cool to ambient temperature. Water (1.33 vol) wasadded and the mixture stirred. The solid was collected by filtration,washed with water (2×0.3 vol), and partially dried on the filter undervacuum. The solid was dried to a constant weight (less than 1%difference) in a vacuum oven at 60° C. with a slight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl as an off-white solid.

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid, Form I, Method A

A slurry of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.HCl (1 eq) in water (10 vol) was stirred at ambient temperature. Asample was taken after stirring for 24 hours. The sample was filtered,and the solid was washed with water (2 times). The solid sample wassubmitted for DSC analysis. When DSC analysis indicated completeconversion to Form I, the solid was collected by filtration, washed withwater (2×1.0 vol), and partially dried on a filter under vacuum. Thesolid was then dried to a constant weight (less than 1% difference) in avacuum oven at 60° C. with a slight N₂ bleed to afford Form I as anoff-white solid (98% yield). ¹H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H),7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m,2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m,2H).

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid, Form I, Method B

A solution of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(1.0 eq) in formic acid (3.0 vol) was heated with stirring to 70±10° C.for 8 hours. The reaction was deemed complete when no more than 1.0% AUCby chromatographic methods of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate)remained. The mixture was allowed to cool to ambient. The solution wasadded to water (6 vol), heated at 50° C., and stirred. The mixture wasthen heated to 70±10° C. until the level of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoatewas no more than 0.8% (AUC). The solid was collected by filtration,washed with water (2×3 vol), and partially dried on the filter undervacuum. The solid was dried to a constant weight (less than 1%difference) in a vacuum oven at 60° C. with a slight N₂ bleed to affordCompound 1 in Form I as an off-white solid.

Preparation of Compound 1, Solvate Form A from Compound 1, Form I

Compound 1, Form I (approximately 30 mg) was slurried in 500 μL of anappropriate solvent (for example, methanol, ethanol, acetone,2-propanol, acetonitrile, tetrahydrofuran, methyl acetate, 2-butanone,ethyl formate, and -methyl tetrahydrofuran) for two days. The slurry wasthen filtered centrifugally or under vacuum and was left to dry atambient temperature overnight to yield Compound 1, Solvate Form A.

The DSC trace of Compound 1, Acetone Solvate Form A is shown in FIG. 13,showing two phase transitions. The melting point for Compound 1, AcetoneSolvate Form A occurs at about 188° C. and 205° C.

An actual X-ray powder diffraction pattern of Compound 1, Solvate Form Ais shown in FIG. 1. Table 2 lists the actual peaks for FIG. 1 indescending order of relative intensity. Table 2.

TABLE 2 2θ Angle [degrees] Relative Intensity [%] 21.70 100.0 8.98 65.511.04 57.4 18.16 55.9 23.06 55.4 20.63 53.1 22.22 50.2 18.57 49.1 16.6647.2 19.86 35.0

Conformational depictions of Compound 1, Acetone Solvate Form A based onsingle crystal X-ray analysis are shown in FIGS. 15-18. FIG. 15 shows aconformational image of Compound 1, Acetone Solvate Form A, based onsingle crystal X-ray analysis. FIG. 16 provides a conformational imageof Compound 1, Acetone Solvate Form A as a dimer showing hydrogenbonding between the carboxylic acid groups based on single X-ray crystalanalysis. FIG. 17 provides a conformational image of a tetramer ofCompound 1, Acetone Solvate Form A. FIG. 18 provides a confirmation ofCompound 1, Acetone Solvate Form A, based on single crystal X-rayanalysis. The stoichiometry between Compound 1, Solvate Form A andacetone is approximately 4.4:1 (4.48:1 calculated from ¹H NMR; 4.38:1from X-ray). The crystal structure reveals a packing of the moleculeswhere there are two voids or pockets per unit cell, or 1 void per hostmolecule. In the acetone solvate, approximately 92 percent of voids areoccupied by acetone molecules. Compound 1, Solvate Form A is amonoclinic P2₁/n space group with the following unit cell dimensions:a=16.5235(10) Å, b=12.7425(8) Å, c=20.5512 (13) Å, α=90°, β=103.736(4)°,γ=90°, V=4203.3(5) Å³, =4. The density of Compound 1 in Compound 1,Solvate Form A calculated from structural data is 1.430/cm³ at 100 K.

A solid state ¹³C NMR spectrum of Compound 1, Acetone Solvate Form A isshown in FIG. 22. Table 3 provides chemical shifts of the relevantpeaks.

TABLE 3 Compound 1, Acetone Solvate Form A ¹³C Chem. Shifts Peak # F1[ppm] Intensity 1 202.8 6.05 2 173.3 62.66 3 171.9 20.53 4 153.5 28.41 5150.9 21.68 6 150.1 19.49 7 143.2 45.74 8 142.3 42.68 9 140.1 37.16 10136.6 26.82 11 135.9 30.1 12 134.6 39.39 13 133.2 23.18 14 131.0 60.9215 128.5 84.58 16 116.0 34.64 17 114.2 23.85 18 112.4 25.3 19 110.924.12 20 107.8 18.21 21 32.0 54.41 22 22.2 20.78 23 18.8 100

A solid state ¹⁹F NMR spectrum of Compound 1, Acetone Solvate Form A isshown in FIG. 23. Peaks with an asterisk denote spinning side bands.Table 4 provides chemical shifts of the relevant peaks.

TABLE 4 Compound 1, Acetone Solvate Form A ¹⁹F Chem. Shifts Peak # F1[ppm] Intensity 1 −41.6 12.5 2 −46.4 6.77 3 −51.4 9.05Preparation of Compound 1, HCl Salt Form A.

Colorless crystals of Compound 1, HCl Salt Form A was obtained by slowevaporation from a concentrated solution of the HCL salt of compound 1in ethanol. A crystal with dimensions of 0.30×⅕×0.15 mm was selected,cleaned using mineral oil, mounted on a MicroMount, and centered on aBruker APEXII diffractometer. Three batches of 40 frames separated inreciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were obtained and refinedbased on the full data set.

FIG. 19 provides a conformational image of Compound 1, HCl Salt Form Aas a dimer, based on single crystal analysis. FIG. 20 provides a packingdiagram of Compound 1, HCl Salt Form A, based on single crystalanalysis. An X-ray diffraction pattern of Compound 1, HCl Salt Form Acalculated from the crystal structure is shown in FIG. 21. Table 5contains the calculated peaks for FIG. 21 in descending order ofrelative intensity.

TABLE 5 2θ Relative Intensity [degrees] [%] 8.96 100.00 17.51 48.2018.45 34.60 10.33 32.10 16.01 18.90 11.94 18.40 8.14 16.20 10.10 13.9016.55 13.30 9.54 10.10 16.55 13.30AssaysAssays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompoundsMembrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane, and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR, a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hours at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hours at 27° C. to “temperature-correct” ΔF508-CFTR. The cellswere subsequently rinsed three times with Krebs Ringers solution andloaded with the voltage-sensitive dyes. To activate ΔF508-CFTR, 10 μMforskolin and the CFTR potentiator, genistein (20 μM), were added alongwith Cl⁻-free medium to each well. The addition of Cl⁻-free mediumpromoted Cl⁻ efflux in response to ΔF508-CFTR activation, and theresulting membrane depolarization was optically monitored using theFRET-based voltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 seconds, asecond addition of CF-free medium containing 2 to 10 μM forskolin wasadded to activate ΔF508-CFTR. The extracellular Cl⁻ concentrationfollowing both additions was 28 mM, which promoted Cl⁻ efflux inresponse to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

3. Solutions

Bath Solution #1 (in mM): NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts.

Prepared as a 10 mM stock solution in DMSO and stored at −20° C.

Prepared as a 10 mM stock in DMSO and stored at −20° C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hours at 37° C. before culturing at 27° C. for24 hours for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16 to 24hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds

1. Using Chamber Assay

Chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed three times prior torecording. The cAMP- and genistein-mediated I_(SC) in compound-treatedcells was normalized to the 27° C. and 37° C. controls and expressed aspercentage activity. Preincubation of the cells with the correctioncompound significantly increased the cAMP- and genistein-mediated I_(SC)compared to the 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers wereused on the basolateral membrane and permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30minutes after nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

4. Solutions

Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂ (1.2),K₂HPO₄ (2.4), KHPO₄ (0.6),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (10), anddextrose (10). The solution was titrated to pH 7.4 with NaOH.

Apical solution (in mM): Same as basolateral solution with NaCl replacedwith Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16 to 48hrs to correct for the ΔF508-CFTR. To determine the activity ofcorrections compounds, the cells were incubated at 27° C. or 37° C. withand without the compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5 to 6 MΩ when filled with the intracellular solution.Under these recording conditions, the calculated reversal potential forCl⁻ (E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance greater than 20 GΩ and a series resistance less than 15 MΩ.Pulse generation, data acquisition, and analysis were performed using aPC equipped with a Digidata 1320 A/D interface in conjunction withClampex 8 (Axon Instruments Inc.). The bath contained less than 250 μlof saline and was continuously perifused at a rate of 2 ml/min using agravity-driven perfusion system.

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, theabove-described perforated-patch-recording techniques were used tomeasure the current density following 24-hour treatment with thecorrection compounds. To fully activate ΔF508-CFTR, 10 μM forskolin and20 μM genistein were added to the cells. Under our recording conditions,the current density following 24-hr incubation at 27° C. was higher thanthat observed following 24-hour incubation at 37° C. These results areconsistent with the known effects of low-temperature incubation on thedensity of ΔF508-CFTR in the plasma membrane. To determine the effectsof correction compounds on CFTR current density, the cells wereincubated with 10 μM of the test compound for 24 hours at 37° C. and thecurrent density was compared to the 27° C. and 37° C. controls (%activity). Prior to recording, the cells were washed three times withextracellular recording medium to remove any remaining test compound.Preincubation with 10 μM of correction compounds significantly increasedthe cAMP- and genistein-dependent current compared to the 37° C.controls.

8. Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

9. Solutions

Intracellular solution (in mM): Cs-aspartate (90), CsCl (50), MgCl₂ (1),HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted to 7.35 withCsOH).

Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl (150),MgCl₂ (2), CaCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1×pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslips,cultured for 24 to 48 hours at 27° C. before use to test the activity ofpotentiators, and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5 to 8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1 to 2 seconds. Tomaintain ΔF508-CFTR activity during the rapid perifusion, thenonspecific phosphatase inhibitor F⁻ (10 mM NaF) was added to the bathsolution. Under these recording conditions, channel activity remainedconstant throughout the duration of the patch recording (up to 60minutes). Currents produced by positive charge moving from the intra- toextracellular solutions (anions moving in the opposite direction) areshown as positive currents. The pipette potential (V_(p)) was maintainedat 80 mV.

Channel activity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 seconds of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions

Extracellular solution (in mM): NMDG (150), aspartic acid (150), CaCl₂(5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).

Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA (5), TES(10), and Tris base (14) (pH adjusted to 7.35 with HCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1×pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Using the procedures described above, the activity, i.e., EC50s, ofCompound 1 has been measured by the techniques above and is shown inTable 6.

TABLE 6 IC50/EC50 Bins: +++ <= 2.0 < ++ <= 5.0 < + PercentActivityBins: + <= 25.0 < ++ <= 100.0 < +++ Cmpd. No. BinnedEC50BinnedMaxEfficacy 1 +++ +++

OTHER EMBODIMENTS

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the present invention. Oneskilled in the art will readily recognize from such discussion and fromthe accompanying drawings and claims, that various changes,modifications and variations can be made therein without departing fromthe spirit and scope of the invention as defined in the followingclaims.

We claim:
 1. A solid form of3-(6-(I-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid (Compound 1), characterized as Compound 1, Solvate Form A. 2.Compound 1, Solvate Form A of claim 1, wherein Compound 1, Solvate FormA is characterized as a crystalline lattice of Compound 1 containing aplurality of repeating cavities which are empty or at least partiallyoccupied by a solvate.
 3. Compound 1, Solvate Form A of claim 1, whereinCompound 1, Solvate Form A is characterized as a crystalline lattice ofCompound 1 containing a plurality of repeating cavities, wherein aplurality of the cavities are empty.
 4. Compound 1, Solvate Form A ofclaim 1, characterized by one or more peaks at 21.5 to 21.9 degrees, 8.8to 9.2 degrees, and 10.8 to 11.2 degrees in an X-ray powder diffractionobtained using Cu K alpha radiation.
 5. Compound 1, Solvate Form A ofclaim 1, characterized by one or more peaks at 21.5 to 21.9 degrees, 8.8to 9.2 degrees, 10.8 to 11.2 degrees, 18.0 to 18.4 degrees, and 22.9 to23.3 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation.
 6. Compound 1, Solvate Form A of claim 1, characterized byone or more peaks at 21.70, 8.98, 11.04, 18.16, and 23.06 degrees in anX-ray powder diffraction obtained using Cu K alpha radiation. 7.Compound 1, Solvate Form A of claim 1, characterized by a peak at 21.5to 21.9 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation.
 8. Compound 1, Solvate Form A of claim 7, characterized by apeak at 21.70 degrees in an X-ray powder diffraction obtained using Cu Kalpha radiation.
 9. Compound 1, Solvate Form A of claim 7, furthercharacterized by a peak at 8.8 to 9.2 degrees in an X-ray powderdiffraction obtained using Cu K alpha radiation.
 10. Compound 1, SolvateForm A of claim 9, further characterized by a peak at 8.98 degrees in anX-ray powder diffraction obtained using Cu K aloha radiation. 11.Compound 1, Solvate Form A of claim 9, further characterized by a peakat 10.8 to 11.2 degrees in an X-ray powder diffraction obtained using CuK alpha radiation.
 12. Compound 1, Solvate Form A of claim 11, furthercharacterized by a peak at 11.04 in an X-ray powder diffraction obtainedusing Cu K alpha radiation.
 13. Compound 1, Solvate Form A of claim 11,further characterized by a peak at 18.0 to 18.4 degrees in an X-raypowder diffraction obtained using Cu K alpha radiation.
 14. Compound 1,Solvate Form A of claim 13, further characterized by a peak at 18.16degrees in an X-ray powder diffraction obtained using Cu K aloharadiation.
 15. Compound 1, Solvate Form A of claim 13, furthercharacterized by a peak at 22.9 to 23.3 degrees in an X-ray powderdiffraction obtained using Cu K alpha radiation.
 16. Compound 1, SolvateForm A of claim 15, further characterized by a peak at 23.06 degrees inan X-ray powder diffraction obtained using Cu K alpha radiation. 17.Compound 1, Solvate Form A of claim 2, wherein the Compound 1, SolvateForm A is characterized by a diffraction pattern substantially similarto that of FIG.
 1. 18. Compound 1, Solvate Form A of claim 2, whereinthe solvent is selected from the group consisting of an organic solventof sufficient size to fit in the plurality of repeating cavities. 19.Compound 1, Solvate Form A of claim 18, wherein the solvent is selectedfrom the group consisting of methanol, ethanol, acetone, isopropanol,acetonitrile, tetrahydrofuran, methyl acetate, 2-butanone, ethylformate, ethyl acetate, and 2-methyltetrahydrofuran.
 20. Compound 1,Solvate Form A of claim 18, wherein Compound 1, Solvate Form A comprises1 to 10 wt. % solvent as determined by TGA.
 21. Compound 1, Solvate FormA of claim 2, wherein Compound 1, Solvate Form A comprises 2 to 5 wt. %solvent as determined by TGA.
 22. Compound 1, Solvate Form A of claim 2,wherein the melting point is from 185° C. to 190° C.
 23. Compound 1,Solvate Form A of claim 2, having a P2₁/n space group, and the followingunit cell dimensions: a=16.5235 (10) Å α=90° b=12.7425 (8)Å β=103.736(4) 0 c=20.5512 (13)Å γ=90°.
 24. Compound 1, Solvate Form A of claim 1,wherein Compound 1, Solvate Form A has a particle size of 0.1 microns to50 microns.
 25. Compound 1, Solvate Form A of claim 1, wherein Compound1, Solvate Form A has a particle size of 0.1 microns to 20 microns. 26.Compound 1, Solvate Form A of claim 1, wherein Compound 1, Solvate FormA has a particle size of 0.1 microns to 10 microns.
 27. Compound 1,Solvate Form A of claim 1, wherein Compound 1, Solvate Form A has aparticle size of 1.0 microns to 5 microns.
 28. Compound 1, Solvate FormA, wherein Compound 1, Solvate Form A has a particle size D50 of 2.0microns.
 29. A pharmaceutical composition comprising Compound 1, SolvateForm A of claim 1, and a pharmaceutically acceptable carrier.
 30. Thepharmaceutical composition of claim 29 further comprising an additionaltherapeutic agent.
 31. A method of treating a CFTR mediated disease in amammal comprising administering to the mammal an effective amount ofCompound 1, Solvate Form A of claim 1, wherein the CFTR mediated diseaseis selected from chloride channelopathies, COPD, asthma, cysticfibrosis, male infertility caused by congenital bilateral absence of thevas deferens, pancreatitis, pancreatic insufficiency, and idiopathicpancreatitis.
 32. The method of claim 31, wherein the CFTR mediateddisease is cystic fibrosis or COPD.
 33. The method of claim 32, whereinthe CFTR mediated disease is cystic fibrosis.
 34. The method of claim32, wherein said patient has a cystic fibrosis transmembrane receptor(CFTR) with a Δ F508 mutation.
 35. The method of claim 32, wherein saidpatient has a cystic fibrosis transmembrane receptor (CFTR) with a R117Hmutation.
 36. The method of claim 32, wherein said patient has a cysticfibrosis transmembrane receptor (CFTR) with a G551 D mutation.
 37. Themethod of claim 31, wherein the method further comprises administeringan additional therapeutic agent which isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.